Module 6 DNA Quantitation

What does the Degradation Index refer to?

It refers to the data observed when a sample displays a decrease in measured amount for large DNA fragments compared to small DNA fragments.

How can you calculate the degradation index?

Divide the small autosomal quant value by the large autosomal quant value

What may a large autosomal target concentrations indicate?

They may indicate that a sample is degraded.

What does the internal positive control (IPC) do?

enables verification that the polymerase, the assay, and the detection instrumentation are working correctly.

What does the Large autosomal target

used mainly as an indicator of DNA degradation, by comparing the ratio of its quantification result with that of the SA target.

What is a Multi-copy target?

refers to the use of multiple copies of specific DNA sequences for improved detection sensitivity and accuracy in various applications.

What is the Quantifiler Trio kit?

The Quantifiler™ Trio Kit is designed to simultaneously quantify the total amount of amplifiable human DNA and human male DNA in a sample.

What is Real-time PCR?

A technique used to measure the amount of DNA during the amplification process.

What does Real-time PCR use to measure DNA amplification?

A fluorescent dye or probe.

How does fluorescence intensity relate to DNA product in Real-time PCR?

Fluorescence intensity correlates with the amount of DNA product.

What type of measurement does Real-time PCR allow for?

Quantitative measurement of DNA.

What does the slope indicate?

Indicates the PCR amplification efficiency for the assay.

What does the small autosomal target?

primary quantification target for total human genomic DNA.

What does the Y-chromosome target?

allows the quantification of a sample's human male genomic DNA component, and is particularly useful in assessing mixture samples of male and female genomic DNAs.

What does the Y-intercept indicates?

Indicates the expected CT value for a sample with Qty = 1.

What is the primary purpose of quantitating DNA before amplification?

To determine the appropriate amount of DNA template to include in PCR amplification.

What is the goal of including the correct amount of DNA template in PCR amplification?

To avoid off-scale data and associated artifacts.

What is a cycle threshold?

The number of cycles needed to replicate enough DNA or RNA to be detected; a lower Ct value indicates a higher amount of DNA or RNA in the sample.

The intensity of the fluorescence

Increases proportionally to the amount of target DNA present as amplification progresses.

What is a standard curve?

Created by plotting the Ct values of the standard dilutions against their corresponding DNA concentrations.

What is the IPC labeled with?

Internal Positive Control, labeled with a JUN dye with QSY quencher.

What is the human probe labeled with?

Labeled with ABY dye with QSY quencher for Large autosomal and VIC dye with MGB quencher for Small Autosomal.

What is a QSY quencher?

A non-fluorescent quencher that detects target DNA amplification

What is a MGB quencher?

Minor groover binder quencher used in fluorescent probes to detect target DNA sequences.

What is the male probe labeled with?

Labeled with FAM dye with MGB quencher.

What is the passive reference labeled with?

Labeled with mustang purple dye.

What is the TaqMan probe?

A type of molecular probe used in real-time PCR to detect and quantify specific DNA sequences, consisting of a short oligonucleotide with a fluorescent dye and a quencher.

What is the reporter dye?

A fluorescent dye linked to the 5′ end of the TaqMan probe.

What is PCR amplification?

The process where the system performs cycles with temperature changes while monitoring fluorescence signal generated by the probes.

Why will the IPC CT be flagged?

Undetermined or greater than the average of the IPC CT values for all the standards plus the threshold you set in the software HID settings.

What factors affect the degradation index?

Degree of degradation of the large autosomal target DNA and presence of PCR inhibitors.

What does the background assay measure?

Measures the ambient fluorescence within the instrument, capturing the baseline signal that is not related to the sample itself.

What is the pure dye spectra?

Refers to the unique fluorescence profile of each individual dye used in the reaction, allowing accurate differentiation and quantification of signals from different fluorescent dyes.

When is the amplicon measurement in RT-PCR?

Measured during the exponential phase of the amplification process because this is the stage where the amount of PCR product doubles with each cycle.

What is R²

An R² value ≥0.98 indicates a close fit between the standard curve regression line and the individual CT data points of quantification standard reaction.

What does the slop indicates in PCR?

Indicates the PCR amplification efficiency for the assay; a slope of −3.3 indicates 100% amplification efficiency.

What is the acceptable slope range for small autosomal?

−3.0 to −3.6.

What is the acceptable slope range for large autosomal?

−3.1 to −3.7.

What effect does the slope have on the concentration?

More negative slope will overestimate the concentration below 1 ng; more positive slope will underestimate it. Above 1 ng, the more negative slope will underestimate concentration and the more positive slope will overestimate it.

What does having a high Y intercept mean?

More DNA